Water soluble derivatives of taxol

ABSTRACT

Sulfonated 2&#39;-acryloyltaxol and sulfonated 2&#39;-O-acyl acid taxol derivatives are synthesized which have improved water solubility and stability while maintaining bio-activity. In particular, 2&#39;-[(3-sulfo-1-oxopropyl)oxy]taxol sodium salt is synthesized by reacting taxol with acrylic acid, and subsequently reacting the 2&#39;-acryloyltaxol with bisulfite in a Michael reaction. 2&#39;-{[4-((2-sulfoethyl)amino)-l,4-dioxobutyl]oxy} taxol sodium salt and 2&#39;-{[4-((3-sulfopropyl)amino)-1,4-dioxobutyl]oxy} taxol sodium salt are synthesized by reacting 2&#39;-succinyltaxol with the tetrabutylammonium salts of taurine and 3-aminopropyl sulfonic acid, respectively, and subsequently exchanging the ammonium with sodium. Glycol derivatives of 2&#39;-O-acyl acid taxols with improved water solubility are synthesized by reaction of a glycol with 2&#39;-O-acyl acid taxol.

This application is a Divisional of U.S. patent application Ser. No.07/573,371, filed Aug. 28, 1990 now U.S. Pat. No. 5,059,699.

FIELD OF THE INVENTION

The present invention relates to water soluble derivatives of taxol withanti-neoplastic activity, and relates more particularly to sulfonated2'-acryloyltaxol derivatives, 2'-sulfoalkylamino-O-acyl acid taxolderivatives, and 2'ethylene glycol-O-acyl acid taxol derivatives.

BACKGROUND OF THE INVENTION

Taxol is a naturally occurring diterpenoid which has great potential asan anti-cancer drug, and which has shown activity in several tumorsystems. Taxol was first isolated and its structure reported by Wani, etal., in "Plant Anti-Tumor Agents. VI. The Isolation And Structure OfTaxol, A Novel Anti-Leukemic And Anti-Tumor Agent From Taxusbrevifolia," J. Am. Chem. Soc., 1971, 93, 2325. Taxol is found in thestem bark of the Western Yew, Taxus brevifolia, as well as in T. baccataand T. cuspidata.

The biological activity of taxol is related to its effect on celldivision. Taxol promotes formation of microtubules that form the mitoticspindle during cell division. However, taxol prevents depolymerizationof the tubulin forming the microtubules of the mitotic spindle, which isessential for cell division to take place. Thus, taxol causes celldivision to stop. Taxol's mechanism is unique since it promotes theformation of tubulin polymers, whereas other anti-cancer drugs, such asvinblastine and colchicine, prevent microtubule formation.

Extensive testing of taxol has not been performed because taxol is inshort supply and has not yet been successfully synthesized. Preliminarystudies have shown that taxol may have marginal activity in acuteleukemia and melanoma, and some activity has been noted in other tumors.Further, studies by McGuire et al. found taxol to be an active agentagainst drug-refractory ovarian cancer. See "Taxol: A UniqueAntineoplastic Agent With Significant Activity In Advanced OvarianEpithelial Neoplasms," Ann. Int. Med, 1989, 111, 273-279, hereinincorporated by reference. However, due to the low water solubility oftaxol, doses had to be delivered as infusions diluted in aqueousdextrose solutions.

It should be noted that in phase 1 clinical trials, taxol itself did notshow excessive toxic effects, but severe allergic reactions were causedby the emulsifiers administered in conjunction with taxol to compensatefor taxol's low water solubility. In fact, at least one patient's deathwas caused by an allergic reaction induced by the emulsifiers.Therefore, researchers have attempted to create water solublederivatives of taxol which retain their anti-neoplastic and anti-canceractivity.

With reference to FIG. 1, the structure of taxol is illustrated alongwith a ¹ H nuclear magnetic resonance (NMR) spectrum of a taxol sample.The NMR signals are well separated and cover the region from 1.0 to 8.2ppm. For simplicity, the spectrum is divided into three regions: a firstregion between 1.0 and 2.5 ppm formed by strong 3-proton signals of themethyl and acetate groups as well as complex multiplets caused bycertain methylene groups; a second region between 2.5 and 7.0 ppmrepresents the signals observed from most of the protons on the taxaneskeleton and the side chain; a third region between 7.0 and 8.2 ppm isformed by the signals from the aromatic protons of the C-2 benzoate,C-3' phenyl and C-3' benzamide groups. The peaks of the NMR spectrum inFIG. 1 are labeled according to the number of the carbon in the taxolstructure to which the protons inducing the signals are attached.

Magri and Kingston reported on the biological activity of taxolssubstituted at the C-2' and C-7 positions in order to make them morewater soluble. See "Modified Taxols, 4.¹ Synthesis And BiologicalActivity Of Taxols Modified In The Side Chain," Journal Of Naturalproducts vol. 51, no. 2 pp. 298-306, March-April 1988,hereinincorporated by reference. A 2'-(t-butyldimethylsilyl)taxol wassynthesized and found to be essentially inactive; this was taken as anindication of the need for a free hydroxyl group at the 2' position ofthe taxol side chain for biological activity. Further, acyl substituentsat the 2' position in 2'-acetyltaxol and 2',7-diacetyltaxol were readilyhydrolyzed under in vivo conditions, and both showed activity in a cellculture bioassay. The lability of the acyl substituents at the 2'position suggested that 2'-acetyltaxols could serve as pro-drug forms oftaxol. (Generally, a prodrug is a compound which exhibits pharmacologicactivity after biotransformation.)

Magi and Kingston reported that two taxols with increased watersolubility were prepared, 2'-(β-alanyl)taxol: ##STR1## and2'-succinyltaxol: ##STR2## The 2'-(β-alanyl)taxol was found to be activein vivo and in vitro, but was unstable. The 2'-succinyltaxol, preparedby the treatment of taxol with succinic anhydride, had a much diminishedP-388 in vivo activity as compared with taxol. Thus, research effortswere concentrated on other derivatives of taxol which did not sufferfrom instability, or inactivity in vivo or in vitro.

Deutsch et al., in "Synthesis Of Congeners And Prodrugs. 3.¹Water-Soluble Prodrugs Of Taxol With Potent Antitumor Activity," J. Med.Chem. 1989, 32 788-792, herein incorporated by reference, reported thatsalts of 2'-succinyltaxol and 2'-glutaryltaxol had improved antitumoractivities when compared to the free acids. Since these researchersbelieved that salts prepared with different counterions often havesubstantially different properties, a variety of 2' substituted taxolsalts were synthesized and tested. Triethanolamine and N-methylglucaminesalts of the 2' substituted taxol derivatives showed greatly improvedaqueous solubility and had more activity than sodium salts. Further, aseries of 2'-glutaryltaxol salts were found to have higher activity thantheir 2'-succinyltaxol analogs. In particular, the taxol salt resultingfrom the coupling of 2'-glutaryltaxol with3-(dimethylamino)-1-propylamine using N, N'carbonyldiimidazole (CDI),demonstrated good solubility and bioactivity.

In addition to increasing the solubility and bioactivity of taxol, it isdesirable that the taxol derivatives formed have increased stability toprolong their shelf life. It is believed that salts of taxol esters arevery susceptible to base hydrolysis, and water-solubilizing groups, suchas carboxylate salts or amine salts, tend to be basic. Thus, it isdesired that neutral, water-soluble taxol derivatives be synthesizedwhich also have improved or the equivalent activity to taxol. Organicsulfonate salts tend to be neutral or only slightly basic, andtherefore, sulfonate salts of taxol esters should have improvedstability. Further, due to the difficulties involved in synthesizingcarboxylic and amine salts of taxol esters, it is desirable to find lessexpensive water-soluble taxol derivatives and processes for formingthem.

SUMMARY OF THE INVENTION

The present invention relates to the production of water soluble taxolderivatives, and water soluble sulfonate salts of taxol. In a preferredembodiment, 2'-[(3-sulfo-1-oxopropyl)-oxy] taxol sodium salt is formedby reacting taxol with acrylic acid to form 2'-acryloyltaxol; the2'-acryloyltaxol is then subjected to a Michael reaction with sodiumbisulfite to form the 2'-sulfoethyl ester salt of taxol. In anotherpreferred embodiment, 2'-O-acyl acid taxols, such as 2'-succinyltaxoland 2'-glutaryltaxol, are subjected to a novel reaction with thetetrabutylammonium salt of taurine to form sulfoalkylamine salts of the2'-O-acyl acid taxols. Another preferred embodiment involves thereaction of amino sulfonic acid salts with succinic or glutaricanhydride, and reaction of the product with taxol to formsulfoalkylamine 2'-O-acyl acid taxol derivatives. In a furtherembodiment, ethylene glycol derivatives of 2'-O-acyl acid taxols areformed. These compounds exhibit high water solubility, and demonstrateanti-leukemic, antineoplastic, and/or anti-cancer activity.

Thus, it is a primary object of this invention to produce water-solublederivatives of taxol with high bioactivity and stability.

It is a further object of the present invention to provide a simple andinexpensive process for forming 2'-acryloyltaxols and their sulfonatesalt derivatives.

It is yet another object of the present invention to produce 2'-O-acylacid taxols and their sulfoalkylamine salts.

It is a still further object of the present invention to producesulfoalkylamine derivatives of 2'-O-acyl acid taxols by simple andinexpensive processes.

It is yet a further object of the present invention to producehydroxyalkoxy derivatives of 2'-O-acyl acid taxol.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a representation of the taxol structure and its nuclearmagnetic resonance spectrum with peaks labeled according to the part ofthe taxol structure to which they correspond.

DETAILED DESCRIPTION OF THE INVENTION

Taxol was obtained from the National Cancer Institute. ¹ H-NMR and ¹³C-NMR spectra were made with a Bruker 270SY 270 MHz spectrometer; 2D-NMRwere obtained using a Bruker WP200 200 MHz spectrometer. Chemical shiftsare all recorded in parts per million (ppm) downfield from TMS in ¹H-NMR, and ¹³ C-NMR chemical shifts are based on chloroform's shift at77.0 ppm or on the TMS shift at 0 ppm. Samples were generally recordedwhile in CDCl₃ or CD₃ OD at ambient temperature. Mass spectra wereobtained using a Finnegan-MAT 112 gas chromatograph-mass spectrometerand VG 7070 HF mass spectrometer equipped with data system, FAB source,and EI/CI source. NMR and mass spectroscopy data are most useful instudying taxol and its derivatives, with other methods, such as IR andUV, providing additional structure confirmation information.

Other analytical instruments used included Perkin-Elmer 710B infraredand Perkin-Elmer 330 UV-visible spectrophotometers, and a Perkin-Elmerpolarimeter. HPLC was carried out on an apparatus consisting of a WatersM6000 pump, a Rheodyne injection valve, a Waters Radial-Pak RLM-100 RP-8column, and a Waters 440 UV detector.

2'-ACRYLOYLTAXOLS

2'-[(3-sulfo-1-oxopropyl)oxy] taxol sodium salt was prepared by couplingtaxol with acrylic acid followed by Michael addition of bisulfite ion.Taxol was reacted with the acrylic acid using isobutylchloroformate asthe coupling agent. This produced 2'-acryloyltaxol in 94% yield afterpurification via flash chromatography (silica gel, 1/1dichloromethane/ethyl acetate). Using TLC, the coupling of acrylic acidto taxol was found to be 90% complete in 15 hours at 60° C. Thedisubstituted C-2', C-7 product was not formed after extended reactiontimes. Proton NMR spectra of the 2'-acryloyltaxol showed that the signalfor the C-2' proton was shifted downfield to 5.46 ppm (d, j=3), from the4.73 ppm shift for the C-2' proton in unsubstituted taxol. The downfieldshift is consistent with acylation of the C-2' hydroxyl group. Since thesignal for the C-7 proton at 4.43 ppm was essentially unchanged whencompared with the unsubstituted taxol C-7 proton signal at 4.38 ppm, itwas concluded that no reaction had taken place at the C-7 position. Massspectroscopy indicated a molecular weight of 907 with peaks at m/z 930(MNa⁺) and 908 (MH⁺).

The 2'-acryloyltaxol was then reacted with sodium bisulfite in a Michaeladdition reaction. Sodium bisulfite was used because it is a goodnucleophile, and because it provides suitable pH conditions for thereaction. Proton NMR spectra of the Michael addition reaction productwere contrasted with the spectra of the 2'-acryloyltaxol. The signals inthe NMR spectra of the 2'-acryloyltaxol that are due to the presence ofthe vinyl protons were not present in the spectra of the Michaeladdition product. However, two triplets at 3.14 ppm and 2.93 ppmindicated the presence of the two new methylene groups in the Michaeladdition product. Mass spectroscopy of the Michael reaction productindicated a molecular weight of 1011 with peaks present at m/z 1034(MNa⁺) and 1012 (MH⁺).

The formation of 2'-[(3-sulfo-1-oxopropyl)oxy] taxol sodium salt wasattempted in a one-step reaction by combining taxol with3-hydroxy-3-oxopropyl sulfonic acid in the presence of pyridine and DCC(dicyclohexylcarbodiimide), but no product was obtained. This ispossibly due to inter-molecular attack by the sulfonyl group on thereaction intermediate.

2'-O-ACYL ACID TAXOL DERIVATIVES

2'-{[4-((2-sulfoethyl) amino)-1,4-dioxobutyl]oxy}taxol sodium salt and2'-{[4-((3-sulfopropyl) amino-1,4-dioxobutyl]oxy} taxol sodium salt wereproduced in high yield by coupling 2'-succinyltaxol with taurine(2-aminoethyl sulfonic acid) and 3-aminopropyl sulfonic acidtetrabutylammonium salts, respectively. Note that other quaternaryammonium salts may be used to make the aminoalkyl sulfonic acids organicsolvent soluble. 2'-succinyltaxol was formed by the reaction of succinicanhydride with taxol for two hours at room temperature in pyridine orDMP. In comparison with the NMR spectrum of taxol, the NMR spectrum of2'-succinyltaxol showed a downfield shift of the C-2' proton signal to5.51 ppm, and the succinyl proton caused multiplets centered about 2.6ppm.

The 2'-succinyltaxol was then reacted with taurine tetrabutylammoniumsalt using isobutylchloroformate as the coupling agent.2'-{[4-((2-sulfoethyl)amino)-1,4-dioxobutyl]oxy}taxol tetrabutylammoniumsalt was produced in 100% yield after isolation via flash chromatographyon silica gel using 7/1 dichloromethane/methanol. The reaction was only80% complete in two hours as monitored by TLC; in order to obtain 100%yield, extended reaction times were necessary. The NMR spectrum of thesulfoalkylamine derivative of 2'-succinyltaxol showed new peaks at 3.6ppm and 2.94 ppm for the two methylene groups. The sodium salt of2'-{[4-((2-sulfoethyl)amino)-1,4-dioxobutyl]oxy}taxol was achieved byrunning 2'-{[4-((2-sulfo-ethyl)amino)-1,4-dioxo-butyl]oxy}taxoltetrabutylammonium salt through a Dowex 50 ion exchange column (Na⁺form). An NMR spectrum of the sodium salt showed the absence of signalsfor the tetrabutyl group. Mass spectroscopy of the sodium salt indicateda molecular weight of 1082 by the presence of peaks at m/z 1105 (MNa⁺)and 1083 (MH⁺). 2'-{[4-((3-sulfopropyl)amino)-1,4-dioxobutyl]oxy}taxolsodium salt was prepared by the same method used for thesulfoethylaminotaxol sodium salt; however, the taurine was replaced with3-amino-1-sulfopropionic acid tetrabutylammonium salt. An NMR spectrumconfirmed the synthesis of the 3-sulfopropylamino derivative; new peakswere present at 3.28, 1.98, and 2.87 ppm, representing the threeadditional methylene groups forming the propyl moiety. The sodium saltform of the sulfopropylamino-succinyltaxol derivative was formed bypassing the tetrabutyl-ammonium salt through a Dowex 50 ion exchangecolumn (Na⁺ form). Mass spectroscopy of the sodium salt of thesulfopropylaminosuccinyltaxol derivative indicated a molecular weight of1096 by the presence of peaks at m/z 1119 (MNa⁺) and 1097 (MH⁺) .

It is also contemplated that an amide linkage can be formed between anamino sulfonic acid and an anhydride or diacid, and that the product canbe reacted with taxol to form water soluble 2'-O-acyl acid taxolderivatives. Preferably, the amino sulfonic acid is an organic solventsoluble salt.

Attempts to form 2'-{[4-((2-sulfoethyl)amino)-1,4 -dioxobutyl]oxy}taxolsodium salt directly from 2'-succinyltaxol in a one-step reaction wereunsuccessful. 2'-succinyltaxol was combined with triethanolamine,isobutylchloroformate, tetrahydrofuran (THF), taurine, DMF, and water.However, water, necessary to solubilize taurine, hydrolyzed the mixedanhydride intermediate back to the starting material. When nonaqueousconditions were tried, the reaction still did not succeed because thetaurine did not dissolve in the organic solvents.

2'-{[4-((2-ethanethiol)amino)-1,4-dioxobutyl]oxy}taxol was prepared inlow yield by combining 2'-succinyltaxol with triethylamine,isobutylchloroformate, THF, 2-thioethylamine and dichloromethane.Attempts to oxidize the thiol to the desired sulfonic acid withmeta-chloroperbenzoic acid, MCPBA, and dichloromethane did not yieldappreciable amounts of the desired sulfoalkylamine succinyltaxolderivative.

ETHYLENE GLYCOL DERIVATIVES OF SUCCINYLTAXOL

2'-{[4-((hydroxylethyl)oxy)-1,4-dioxobutyl]oxy}taxol was prepared bycoupling succinyltaxol with ethylene glycol. Thehydroxyethyloxysuccinyltaxol derivative was formed in 83% yield after areaction time of 20 hours at room temperature. Thehydroxyethyloxysuccinyltaxol derivative was made in order to convert thesecondary hydroxyl group at the 2' position in taxol to a primaryhydroxyl group; it is hypothesized that the hydroxyl group in theproduct is more reactive than that of the hydroxyl in taxol, and thatthis will make it possible to make other taxol derivatives under mildconditions. An NMR spectrum of the ethylene glycol derivative showed thepresence of new peaks at 3.7 ppm and 4.1 ppm, which are assigned to thetwo new methylene groups of the hydroxyethyloxy derivative. Massspectroscopy indicated a molecular weight of 997 by the presence ofpeaks at m/z 1020 (MNa⁺) and 998 (M⁺).

2'-γ-AMINOBUTYRYLTAXOL FORMATE

2'-γ-aminobutyryltaxol formate was synthesized by coupling taxol withN-carbobenzyloxy(CBZ)-γ-aminobutyric acid followed by deprotection ofthe amine. Taxol was reacted with N-CBZ-γ-aminobutyric acid usingdicyclohexylcarbodiimide (DCC) as the coupling agent. The resulting2'-NCBZ-γ-aminobutyryl taxol was produced in 75% yield afterpurification via preparative TLC with silica gel and 3/2 hexane/ethylacetate. DCC decomposes to dicyclohexylurea with the addition of water,so the excess reagents used to drive the reaction did not present aproblem; most of the dicyclohexylurea and N-CBZ-γ-aminobutyric acid wereremoved by filtration. Deprotection of the 2'-N-CBZ-γ-amino-butyryltaxolwas effected using 5% Pd/C as a catalyst and formic acid as a hydrogensource. Formic acid provides an active form of hydrogen for removal ofCBZ protecting groups, and the reaction yields the2'-γ-aminobutyryltaxol derivative as a formate salt, which is more watersoluble than the neutral form. NMR confirmed the synthesis of the2'-γ-aminobutyryl taxol formate. However, the compound was unstable inmethanol solution and decomposed back to taxol after a few hours. Thisinstability precluded further consideration of 2'-γ-amino-butyryltaxolformate as a prodrug form of taxol.

WATER SOLUBILITY

Water solubilities for all compounds were determined by the partitioncoefficient between 1-octanol and water. Octanol saturated withdistilled water and distilled water saturated with octanol were used forthe solubility determinations. Partition experiment results showed that2'-[(3-sulfo-1-oxo-propyl)oxy]taxol sodium salt is 210 times more watersoluble than taxol,2'-{[4-((2-sulfoethyl)amino)-1,4-dioxobutyl]oxy}taxol sodium salt is 191times more soluble than taxol, and2'-{[4-((3-sulfopropyl)amino)-1,4-dioxobutyl]oxy}taxol sodium salt is118 times more water soluble than taxol.

EXAMPLES

The following nonlimiting examples provide specific synthesis methodsfor preparing the water soluble taxol derivatives of the presentinvention. All technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art.Other methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present invention.

EXAMPLE 1

Triethylamine, 50 μl and acrylic acid 30 μl were dissolved in 5 ml dryTHF in a 25 ml round-bottom flask under an argon gas atmosphere. Aftercooling the solution to 0° C. in an icebath, 50 μl ofisobutylchloroformate were added, and the reaction mixture was warmed toroom temperature over a 15-minute period. One hundred milligrams oftaxol were added to the reaction mixture, and the solution was stirredat 60° C. for 15 hours, and monitored by TLC with dichloromethane/ethylacetate (2/1). Triethylamine hydrochloride precipitated during thereaction, and was removed by filtration. The solvent was then removed invacuo, and the product was purified via flash chromatography usingsilica gel and 1/1 dichloromethane/ethyl acetate. This yielded 100 mg(94%) of 2'-acryloyltaxol: ##STR3##

The acryloyl moiety on the 2'-acryloyltaxol is a good Michael acceptordue to the electrophilic β alkene carbon atom which is subject tonucleophilic attack. Thus reaction of 2'-acryloyltaxol with suitablenucleophiles will result in Michael addition at the 2' position. An 85mg quantity of the 2'-acryloyltaxol was dissolved in about 3 ml ofdistilled isopropanol, and 84 mg of sodium meta-bisulfite were dissolvedin about 1 ml of distilled water. The two solutions were mixed together,and the reaction mixture stirred at 60° C. for about 15 hours. TLC with10/1 dichloromethane/methanol was used to monitor the reaction. Thesolvents were then removed under vacuum, and water was removed byazeotroping with acetonitrile. Flash chromatography with 2/1dichloromethane/isopropanol was used to purify the product. A yield of83.5 mg (83.5%) of 2'-[(3-sulfo-1-oxopropyl)oxy]taxol sodium saltresulted: ##STR4## NMR, MS, UV, and IR (KBr) were performed on samplesof the product, and optical rotation, and melting point were determined,with the characterization data and NMR data presented in Tables 1 and 2below.

                  TABLE 1                                                         ______________________________________                                        Characterization Data For                                                     2'-[(3-sulfo-1-oxopropyl)oxy]taxol sodium salt                                ______________________________________                                        m.p.         175-176° C.                                               [α].sup.20 .sub.D                                                                    -30° (0.0012, MeOH)                                       IR (KBr):    3500, 2950, 1760, 1730, 1660, 1380, 1250, - 1190, 1100, 800                   cm.sup.-1                                                        UV λ.sup.MeOH max :                                                                 279 nm (ε 579), 270 nm (ε 869), 228 nm                        (ε 15072)                                                MS (FAB):    1034 (MNa.sup.+), 1012 (MH.sup.+)                                ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        NMR Data For                                                                  2'-[(3-sulfo-1-oxopropyl)oxy]                                                 taxol sodium salt                                                                      .sup.1 H Shift (ppm from TMS)                                                                   .sup.13 C Shift                                    Postion  Coupling (hertz)  (ppm from TMS)                                     ______________________________________                                        C-1                        *                                                  C-2      6.2 (d,7)         75                                                 C-3      3.82 (d,7)        45.8                                               C-4                        80.5                                               C-5      5.0 (d,9)         84                                                 C-6      2.48 m            35.2                                               C-7      4.35 m            76                                                 C-8                        57.9                                               C-9                        203.8                                              C-10     6.45 s            70.8                                               C-11                       131                                                C-12                       141                                                C-13     6.09 (t,8)        75.4                                               C-14     2.48 m            35.8                                               C-15                       43                                                 C-16     1.15 s            25.9                                               C-17     1.17 s            19.8                                               C-18     1.95 s            13.8                                               C-19     1.67 s            9.4                                                C-20     4.21 s            70.8                                               C-1'                       171                                                C-2'     5.45 (d,3)        74                                                 C-3'     5.84 (d,7)        53.1                                               N-H      7.26 (t,9)                                                           CH.sub.3 (OAc)                                                                         2.2 s             21                                                 CH.sub.3 (OAc)                                                                         2.4 s             21.9                                               Bz       7.4-8.1 m         126.8-138.1                                        CO(OAc)                    168.4                                              CO(OAc)                    169.9                                              CO(OBz)                    166.2                                              CO(NBz)                    168.2                                              C-1"                       170.2                                              C-2"     2.93 (t 8)        29.2                                               C-3"     3.14 (t 8)        63.2                                               ______________________________________                                         *under CHC1.sub.3 signal                                                 

Note that it is anticipated that the acrylic acid used may be replacedwith other members of the acrylic acid family which are also goodMichael acceptors, and that the salt-forming moiety may be anotheralkaline metal, or an ammonio group, such as a tetrabutylammonium group.It is also envisioned that the salt forming moiety may be replaced withH. Biological testing of 2'-[(3-sulfo-1- oxopropyl)oxy]taxol sodium saltdemonstrated that the compound is bioactive in addition to havingimproved water solubility.

EXAMPLE 2

A 206 mg quantity of taxol was combined with 2.9 mg of4-dimethylaminopyridine (DMAP) and 49 mg of succinic anhydride in a 25ml flask equipped with a magnetic stirrer. A 2.0 ml quantity of drypyridine was added, and the solution was stirred at room temperature for2.5 hours. Several milliters of water were then added to produce a whiteprecipitate in an opaque suspension. Several milliters ofdichloromethane were then added to extract the products. Addition of 1ml of concentrated HCl caused the white aqueous suspension to disappear.Sodium sulphate was used to dry the dichloromethane layer, which wasthen filtered and evaporated. TLC with 7/1 CH₂ Cl₂ /MeOH indicated onlya trace of pyridine remaining. The remaining pyridine was removed by thecyclical addition of heptane followed by evaporation; this yielded 218mg of succinyltaxol, representing a 96.6% yield. Proton NMR of theproduct matched values given in the literature. The structure was alsoconfirmed using 2D-NMR HOMO COSY (homonuclear correlation spectroscopy).

Taurine, H₂ NCH₂ CH₂ SO₃ H, is a highly polar compound which isessentially insoluble in organic solvents such as chloroform. Taurinederivatives of organic acids have been made in the past by treating theacid chloride with taurine under Schotten-Baumann conditions (i.e., inbasic aqueous or aqueous-ethanolic solution). This method wasunacceptable for taxol because it is readily hydrolyzed in base, andwould thus decompose under the reaction conditions. In order to overcomethis problem, a new method was developed which involved the addition oftaurine to tetrabutyl-ammonium hydroxide, followed by removal ofunreacted materials and evaporation. This yielded the tetrabutylammoniumsalt of taurine instead of the sodium salt used in the prior art. Thetetrabutylammonium salt of taurine is soluble in organic solvents, suchas dichloromethane. Thus, 2'-succinyltaxol in THF and triethylamine canbe reacted with isobutylchloroformate and taurine tetrabutylammoniumsalt to form the tetrabutylammonium salt of the taxol taurinederivative. Note that the intermediate is a mixed anhydride, whichhydrolyzes back to the starting compound in the presence of water.

A minimum volume of distilled water was used to dissolve 250 mg taurinein a flask, and 1 ml of aqueous tetrabutylammonium hydroxide was addedto the solution. The solution was stirred at room temperature for onehour, and then evaporated to dryness. The dry product was dissolved indry THF (about 15 ml), filtered, and the filtrate was evaporated untildry. The dried product was then redissolved in 2 ml of dried THF.

PREPARATION OF 2'-{[4-((2-SULFOETHYL)AMINO)-1,4-DIOXOBUTYL]OXY}TAXOLTETRABUTYLAMMONIUM SALT

A solution of 2'-succinyltaxol, formed by dissolving 122 mg of2'-succinyltaxol in about 4 ml of dried THF and 50 μl of triethylamine,was cooled to about 0° C. The solution was then combined with 50 μl ofisobutylchloroformate, the reaction mixture was warmed to roomtemperature over a 15-minute period, and 0.5 ml of taurinetetrabutylammonium salt in THF solution (equivalent to 91 mg of taurinetetrabutylammonium salt) were added. Following the addition of thetaurine tetrabutyl-ammonium salt, the reaction mixture was stirred atroom temperature for 5 hours, and the reaction was monitored by TLC with2/1 EtOAc/MeOH. The reaction mixture was then filtered, and the solventswere evaporated. Purification by flash chromatography using silica gel(300×15 mm bed, 7/1 CH₂ Cl₂ /MeOH) yielded 168 mg (100%) of2'-{[4-((2-sulfoethyl)amino)-1,4-dioxobutyl]oxy}taxol tetrabutylammoniumsalt.

The tetrabutylammonium salt was converted to the sodium salt by placing160 mg of the tetrabutylammonium salt in a beaker with Dowex 50 ionexchange resin in the Na⁺ form (about 3 ml of resin in 3 ml of deionizedwater). After stirring the mixture at room temperature for 1.5 hours,the mixture was then passed through a small resin column which contained2 ml of resin in the Na⁺ form, using deionized water as the solvent.

The solution was azeotroped with acetonitrile to yield 122 mg (91.7%) of2'-{[4-((2-sulfoethyl) amino)-1,4-dioxo-butyl]oxy}taxol sodium salt:##STR5##

Characterization data are presented below in Table 3, and NMR chemicalshift data are presented in Table 4 below.

                  TABLE 3                                                         ______________________________________                                        Characterization Data For                                                     2'-{[4-((2-Sulfoethyl)Amino)-1,                                               4-Dioxobutyl]Oxy}taxol Sodium Salt                                            ______________________________________                                        m.p.         174-175° C.                                               [α].sup.20 .sub.D                                                                    -29.8° (0.0055, MeOH)                                     IR (KBr):    3450, 3000, 1760, 1730, 1660, 1560, 1400,                                     1260, 1190, 1050 cm.sup.-1                                       UV λ.sup.MeOH max :                                                                 279 nm (ε 649), 271 nm (ε 8920), 228 nm                       (ε 12824)                                                MS (FAB):    1105 (MNa.sup.+), 1083 (MH.sup.+)                                ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        NMR Data For                                                                  2'-((4-((2-Sulfoethyl)Amino)-1,                                               4-Dioxobutyl)Oxy)taxol Sodium Salt                                                     .sup.1 H Shift (ppm from TMS)                                                                   .sup.13 C Shift                                    Position Coupling (hertz)  (ppm from TMS)                                     ______________________________________                                        C-1                        79                                                 C-2      5.66 (d,7)        76.6                                               C-3      3.8 (d,7)         47.2                                               C-4                        81.6                                               C-5      5.02 (d,9)        85.4                                               C-6      2.52 m            36                                                 C-7      4.35 m            77.3                                               C-8                        58.8                                               C-9                        204.8                                              C-10     6.43 s            72.8                                               C-11                       132.6                                              C-12                       142.2                                              C-13     6.05 (t,8)        75.9                                               C-14     2.14 m            36.2                                               C-15                       44.1                                               C-16     1.18 s            26.8                                               C-17     1.18 s            21                                                 C-18     1.94 s            14.9                                               C-19     1.67 s            10.2                                               C-20     4.23              72                                                 C-1'                       173.4                                              C-2'     5.46 (d,7)        75.8                                               C-3'     5.8 (dd 7,7)      55                                                 N-H      7.27 (t,7)                                                           CH.sub.3 (OAc)                                                                         2.2 s             22.2                                               CH.sub.3 (OAc)                                                                         2.4 s             23.3                                               Bz       7.4-8.1 m         126.8-138.1                                        CO(OAc)                    170.2                                              CO(OAc)                    170.2                                              CO(OBz)                    167.2                                              CO(NBz)                    171.2                                              C-1"                       173.1                                              C-2"     2.72 m            30                                                 C-3"     2.52 m            30                                                 C-4"                       173.1                                              C-1'"    3.58 m            47                                                 C-2'"    2.96 m            51                                                 N-H      3.58 (t,7)                                                           ______________________________________                                    

Note that the tetrabutylammonium salt of taurine may easily be reactedwith other 2'-O-acyl acid taxols, such as 2'-glutaryltaxol.2'-glutaryltaxol can be formed easily by substituting glutaric anhydridefor succinic anhydride. It is believed that other members of the oxalicacid series and other anhydrides may react with taxol more or lessequivalently to the compounds specifically disclosed. Note that, in someinstances, 2'-glutaryltaxol may be preferred to the use of other2'-O-acyl acid taxols. Further, it is contemplated that the salt formingmoiety may be replaced with H or another alkaline or alkaline earthmetal.

EXAMPLE 3

A solution of 280 mg 3-amino-1-sulfopropionic acid in distilled waterwas formed, and 1 ml tetrabutylammonium hydroxide was added. Thesolution was stirred at 60° C. for one hour, and then evaporated todryness. The products were dissolved in about 15 ml THF and excess3-amino-1-sulfo-propionic acid was removed by filtration. The filtratewas evaporated, and redissolved in 2 ml dried THF for subsequentreaction. A solution of 130 mg 2'-succinyltaxol and 50 μl oftriethylamine in 4 ml of dry THF was formed, and the solution was cooleddown to 0° C. A 50 μl aliquot of isobutylchloroformate was added to thereaction mixture, and the solution was warmed to room temperature inabout 15 minutes. This was followed by the addition of 0.6 ml of3-amino-1-sulfopropionic acid tetrabutylammonium salt in THF solution(equivalent to 108 mg of 3-amino-1-sulfopropionic acidtetrabutylammonium salt). The reaction mixture was stirred at roomtemperature for three hours, and reaction progress was monitored by TLCwith 4/1 ethyl acetate/methanol. The reaction solution was then filteredand evaporated, with the product being purified by flash chromatographyusing silica gel (300 mm× 15 mm bed with a 10/1 dichloromethane/methanoleluent). A yield of 128 mg (71.2%) of the homogenous tetrabutylammoniunsalt of taxol resulted.

The tetrabutylammonium salt was converted to the sodium salt by placing120 mg of 2'-{[4-((3-sulfopropyl)amino)-1,4-dioxobutyl]oxy}taxoltetrabutylammoniun salt in a beaker with Dowex 50 ion exchange resin inthe Na⁺ form (approximately 3 ml of resin per 3 ml deionized water). Themixture was stirred at room temperature for about 1.5 hours, and thenpassed through a resin column which contained 2 ml of resin in the Na⁺form, and using deionized water as a solvent. The solution wasazeotroped with acetonitrile and yielded 84 mg (79.3%) of2'-{[4-((3-sulfopropyl)-amino)-1,4-dioxobutyl]oxy}taxol sodium salt:##STR6##

Characterization data for this compound is presented in Table 5, and NMRchemical shift data is presented in Table 6 below.

                  TABLE 5                                                         ______________________________________                                        Characterization Data For                                                     2'-{[4-((3-Sulfopropyl)Amino)-1,                                              4-Dioxobutyl]oxy}taxol Sodium Salt                                            ______________________________________                                        m.p.         168-169° C.                                               [α].sup.20 .sub.D                                                                    -29° (0.001, MeOH)                                        IR (KBr):    3480, 3000, 1760, 1740, 1660, 1550, 1400,                                     1260, 1050 cm.sup.-1                                             UV λ.sup.MeOH max :                                                                 279 nm (ε 974), 271 nm (ε 1240), 228 nm                       (ε 12719)                                                MS (FAB):    1119 (MNa.sup.+), 1097 (MH.sup.+)                                ______________________________________                                    

                  TABLE 6                                                         ______________________________________                                        NMR Data For                                                                  2'-{[4-((3-Sulfoproyl)Amino)-1,                                               4-Dioxobutyl]oxy}taxol Sodium Salt                                                     .sup.1 H Shift (ppm from TMS)                                                                   .sup.13 C Shift                                    Position Coupling (hertz)  (ppm from TMS)                                     ______________________________________                                        C-1                        *                                                  C-2      5.63 (d,7)        74.8                                               C-3      3.8 (d,7)         46                                                 C-4                        80.8                                               C-5      4.99 (d,9)        84.2                                               C-6      2.5 m             34.7                                               C-7      4.34 m            75.9                                               C-8                        57.5                                               C-9                        204.2                                              C-10     6.44 s            71.3                                               C-11                       131.6                                              C-12                       141.2                                              C-13     6.05 (t,8)        75.2                                               C-14     2.14 m            35.7                                               C-15                       42.8                                               C-16     1.16 s            25.6                                               C-17     1.16 s            19.4                                               C-18     1.93 s            13.6                                               C-19     1.67 s            8.9                                                C-20     4.21 s            70.8                                               C-1'                       172                                                C-2'     5.44 (d,7)        74.2                                               C-3'     5.79 (dd 7,7)     53.6                                               N-H      7.25 (t,7)                                                           CH.sub.3 (OAc)                                                                         2.2 s             20.9                                               CH.sub.3 (OAc)                                                                         2.4 s             21.6                                               Bz       7.4-8.1 m         126.8-138.1                                        CO(OAc)                    169                                                CO(OAc)                    170.2                                              CO(OBz)                    166.4                                              CO(NBz)                    170.2                                              C-1"                       171.9                                              C-2"     2.75 (t,7)        29                                                 C-3"     2.54 (t,7)        29.8                                               C-4"                       171.9                                              C-1'"    3.25 m            37.9                                               C-2'"    1.98 m            28.3                                               C-3'"    2.85 (t,7)        **                                                 ______________________________________                                         *under CHC1.sub.3 signal                                                      **under MeOH signal                                                      

Note, it is envisioned that sodium can be replaced with H or any othersalt forming moiety such as other alkaline or alkaline earth metals, andammonio groups.

EXAMPLE 4

A solution of 26 mg 2'-succinyltaxol and 20 μl of triethylamine in 2 mlof dried THF was prepared under argon gas atmosphere, and the solutionwas cooled to 1° C. A 10 μl aliquot of isobutylchloroformate was addedto the solution, and the reaction mixture was warmed to room temperaturein about 15 minutes. Following the warming step, 5 μl ethylene glycolwere added, and the reaction mixture was stirred at room temperature for15 hours, with the reaction progress monitored by TLC with 1:1dichloromethane/ethyl acetate. The reaction was stopped by filtering theprecipitate, and evaporating the solvent. Crude products were purifiedby preparative TLC (1:3 dichloromethane/ethyl acetate), yielding 25 mg(83.3%) of 2'-{[4-((2-hydroxyethyl)oxy)-1,4-dioxobutyl ]oxy}taxol:##STR7##

Characterization data are presented in Table 7 and NMR chemical shiftdata are presented in Table 8 below.

                  TABLE 7                                                         ______________________________________                                        Characterization Data For                                                     2'-{[4-((2-hydroxyethyl)oxy)-1,                                               4-dioxbutyl]oxy}taxol                                                         ______________________________________                                        m.p.         164-165° C.                                               [α].sup.20 .sub.D                                                                    -32.5° (0.002, MeOH)                                      IR (KBr):    3500, 2950, 1760, 1740, 1660, 1390, 1260,                                     1160, 1080, 1040 cm.sup.-1                                       UV λ.sup.MeOH max :                                                                 279 nm (ε 609), 272 nm (ε 831), 228 nm                        (ε 14404)                                                MS (FAB):    1020 (MNa.sup.+), 998 (MH.sup.+)                                 ______________________________________                                    

                  TABLE 8                                                         ______________________________________                                        NMR Data For                                                                  2'-{[4-((2-hydroxyethyl)oxy)-1,                                               4-dioxobutl]oxy}taxol                                                                  .sup.1 H Shift (ppm from TMS)                                                                   .sup.13 C Shift                                    Position Coupling (hertz)  (ppm from TMS)                                     ______________________________________                                        C-1                        79.1                                               C-2      5.7 (d,7)         75.8                                               C-3      3.8 (d,7)         45.8                                               C-4                        81                                                 C-5      4.95 (d,9)        84.3                                               C-6      2.56 m            35.6                                               C-7      4.43 m            75.8                                               C-8                        58.2                                               C-9                        204                                                C-10     6.29 s            72.1                                               C-11                       132                                                C-12                       142.3                                              C-13     6.23 (t,8)        75.8                                               C-14     2.42 m            35.6                                               C-15                       43.2                                               C-16     1.23 s            26.8                                               C-17     1.15 s            20.5                                               C-18     1.94 s            14.3                                               C-19     1.70 s            9.8                                                C-20     4.19 (d,8)        72.1                                               C-1'                       172.2                                              C-2'     5.48 (d,3)        74.3                                               C-3'     5.97 (dd 3,9)     52.9                                               N-H      7.14 (d,9)                                                           CH.sub.3 (OAc)                                                                         2.25 s            22.1                                               CH.sub.3 (OAc)                                                                         2.45 s            22.8                                               Bz       7.4-8.1 m         126.8-138.1                                        CO(OAc)                    168                                                CO(OAc)                    169.9                                              CO(OBz)                    167                                                CO(NBz)                    167.3                                              C-1"                       171                                                C-2"     2.65 m            29                                                 C-3"     2.78 m            29                                                 C-4"                       171                                                C-1'"    3.7 (t,7)         66.2                                               C-2'"    4.1 m             61                                                 ______________________________________                                    

EXAMPLE 5

To a 10 ml flask, 20 mg taxol, 40 mg of dicyclohexyl-carbodiimide, and20 mg of N-carbobenzyl-γ-aminobutyric acid were added. The reactantswere dissolved in 4 ml of dry acetonitrile (dry acetonitrile wasobtained by passing acetonitrile through activated alumina). Afterstirring the reaction mixture at room temperature for 30 hours, thesolution was filtered to remove precipitated dicyclohexylurea. Thesolvent was then removed under vacuum, and the crude products wereseparated by preparative TLC with 45:55 hexane/ethyl acetate. Thisyielded 19.1 mg (75.9%) of pure 2'-N-CBZ-γ-aminobutyryltaxol.

2'-γ-aminobutyl-yltaxol formate was synthesized by the addition of 6 mgof 2'-N-CBZ-γ-aminobutyryltaxol to 1.5 ml of methanol. Upon thedissolution of the CBZ-taxol derivative, 1 ml of formic acid was addedto form a 40% formic acid/methanol solution. The reaction was carriedout by adding 5 mg of 5% of Pd/C to the solution, and stirring it atroom temperature for 26 hours. The reaction was stopped by filtering offthe Pd/C, and drying the filtrate under vacuum. This yielded2'-γ-amino-butyryltaxol formate: ##STR8##

After a few hours, proton NMR and TLC with 2:1:0.02dichloromethane/ethylacetate/methanol showed that the2'-γ-aminobutyryltaxol formate had decomposed back to taxol.

EXAMPLE 6

Taxol's water solubility was determined by dissolving 1.6 mg of taxol in10 ml of distilled water saturated with 1-octanol in a 60 ml separatoryfunnel, and 10 ml of 1-octanol saturated with distilled water was thenadded. The funnel was shaken, and allowed to stand for about 30 minutesuntil the organic and aqueous phases separated. UV absorptionmeasurements at 228 nm were made of the aqueous layer and/or octanollayer, with the octanol layer being diluted 5 times before measurement.

Following the same procedure as above, 0.8 mg of2'-[(3-sulfo-1-oxopropyl)oxy]taxol sodium salt, 0.8 mg of2'-{[4-((2-sulfoethyl)amino)-1,4-dioxybutyl]oxy}taxol sodium salt and0.7 mg of 2'-{[4-((3-sulfopropyl)amino)-1,4-dioxy-butyl]oxy}taxol sodiumsalt had their water solubilities determined relative to taxol; theresults are presented below in Table 9.

                  TABLE 9                                                         ______________________________________                                        TAXOL DERIVATIVE WATER                                                        SOLUBILITIES RELATIVE TO TAXOL                                                Compound             Relative Solubility                                      ______________________________________                                        Taxol                 1                                                       2'-[(3-sulfo-1-oxopropyl)oxy]taxol                                                                 210                                                      sodium salt                                                                   2'-{[4-((2-sulfoethyl)amino)-1,4-di-                                                               191                                                      oxybutyl]oxy}taxol sodium salt                                                2'-{[4-((3-sulfopropyl)amino)-1,4-di-                                                              118                                                      oxybutyl]oxy}taxol sodium salt                                                ______________________________________                                    

Table 9 indicates that the 2'-acryloyltaxol derivative had the highestwater solubility, and is 210 times more water soluble than taxol. Notethat the taurine 2'-succinyltaxol derivative has a much greater watersolubility than the 3-amino-1-sulfopropionic acid derivative of02'-succinyltaxol; however, both compounds have solubilities more than100 times greater than taxol. The decreased solubility for the3-amino-1-sulfopropionic acid derivative of 2'-succinyltaxol is probablydue to the increased alkyl chain length.

Thus, the present invention discloses new taxol derivatives withincreased water solubility in comparison to underivatized taxol, andwhich are stable for longer periods of time than certain previousderivatives of taxol which had increased water solubilities. Thesecompounds are produced by new processes that result in high yields ofessentially pure compounds. Characterization data and NMR studiesconfirm the structure and properties of the taxol derivatives of thepresent invention. In addition to having high water solubilities andimproved stability, these compounds retain their bioactivity andusefulness as antineoplastic, anti-leukemic and anti-cancer prodrugs.

Contemplated equivalents of the water soluble taxol derivatives of thepresent invention include 2'-acryloyl and 2'-O-acyl acid derivatives oftaxol which have one or more side chain or ring substituents substitutedwith a non-interfering group (e.g., a substituted group which does notseriously alter the desirable properties of the taxol derivatives of thepresent invention), such as but not limited to, substitution of --H,--OH, --OR, --NR, --Ar, or ═O for another non-interfering moiety.

From the above teachings, it is apparent that many modifications andvariations of the present invention are possible. It is therefore to beunderstood that the invention may be practiced otherwise than asspecifically described.

We claim:
 1. A process, comprising the reaction of a 2'-O-acyl acidtaxol with a glycol.